Li et al. Biotechnol Biofuels (2017) 10:170 DOI 10.1186/s13068-017-0825-x Biotechnology for Biofuels RESEARCH Open Access Trichoderma reesei complete genome sequence, repeat‑induced point mutation, and partitioning of CAZyme gene clusters Wan‑Chen Li1,2,3†, Chien‑Hao Huang3,4†, Chia‑Ling Chen3, Yu‑Chien Chuang3, Shu‑Yun Tung3 and Ting‑Fang Wang1,3* Abstract Background: Trichoderma reesei (Ascomycota, Pezizomycotina) QM6a is a model fungus for a broad spectrum of physiological phenomena, including plant cell wall degradation, industrial production of enzymes, light responses, conidiation, sexual development, polyketide biosynthesis, and plant–fungal interactions. The genomes of QM6a and its high enzyme-producing mutants have been sequenced by second-generation-sequencing methods and are pub‑ licly available from the Joint Genome Institute. While these genome sequences have ofered useful information for genomic and transcriptomic studies, their limitations and especially their short read lengths make them poorly suited for some particular biological problems, including assembly, genome-wide determination of chromosome architec‑ ture, and genetic modifcation or engineering. Results: We integrated Pacifc Biosciences and Illumina sequencing platforms for the highest-quality genome assem‑ bly yet achieved, revealing seven telomere-to-telomere chromosomes (34,922,528 bp; 10877 genes) with 1630 newly predicted genes and >1.5 Mb of new sequences. Most new sequences are located on AT-rich blocks, including 7 centromeres, 14 subtelomeres, and 2329 interspersed AT-rich blocks. The seven QM6a centromeres separately consist of 24 conserved repeats and 37 putative centromere-encoded genes. These fndings open up a new perspective for future centromere and chromosome architecture studies. Next, we demonstrate that sexual crossing readily induced cytosine-to-thymine point mutations on both tandem and unlinked duplicated sequences. We also show by bioin‑ formatic analysis that T. reesei has evolved a robust repeat-induced point mutation (RIP) system to accumulate AT-rich sequences, with longer AT-rich blocks having more RIP mutations. The widespread distribution of AT-rich blocks correlates genome-wide partitions with gene clusters, explaining why clustering of genes has been reported to not infuence gene expression in T. reesei. Conclusion: Compartmentation of ancestral gene clusters by AT-rich blocks might promote fexibilities that are evo‑ lutionarily advantageous in this fungus’ soil habitats and other natural environments. Our analyses, together with the complete genome sequence, provide a better blueprint for biotechnological and industrial applications. Keywords: Trichoderma reesei, Complete genome sequence, Repeat-induced point mutation, AT-rich block, Centromere, Centromere-encoded gene, CAZyme gene cluster Background caps, rotting wood, and soil. Trichoderma reesei (syn. Trichoderma is a fungal genus found in many diferent Hypocrea jecorina) is a widely used model organism natural habitats including—among others—mushroom for plant cell wall degradation and industrial enzyme production. Te natural strain QM6a (ATCC13631) *Correspondence: [email protected] was frst isolated from the Solomon Islands during the †Wan-Chen Li, Chien-Hao Huang contributed equally to this work Second World War [1]. High enzyme producers (e.g., 3 Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan QM9414 and RUT-C30) were artifcially generated from Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Biotechnol Biofuels (2017) 10:170 Page 2 of 20 QM6a for industrial applications over the last 70 years had been applied to close the gaps between the QM6a- [2–6]. v2.0 scafolds. Te HiC draft genome revealed seven Trichoderma reesei undergoes a heterothallic repro- superscafolds and four short contigs [17]. Druzhinina ductive cycle and generates fruiting bodies (stromata) et al. [18] then applied the QM6a-HiC draft to annotate containing linear asci with 16 ascospores [7]. Tese 16 9151 (not 9194) predicted genes. A third of the putative ascospores are generated via meiosis and two rounds CAZyme genes occurred in loose clusters that also con- of postmeiotic mitosis [8]. When placed under favora- tained a high number of genes encoding small-secreted ble conditions, ascospores germinate to form vegetative cysteine-rich proteins (SSCPs). Five CAZyme gene clus- mycelia and produce asexual spores (i.e., conidia). Sex- ters are located close to chromosomal ends. Tese sub- ual development of the T. reesei CBS999.97 wild-isolate telomeric areas are also enriched in genes involved in strain produced two haploid strains, CBS999.97(MAT1-1, conidiation, iron scavenging, and interactions with other F/X) and CBS999.97(MAT1-2, M/33). Te two ances- fungi, such as secreted protease genes, amino acid trans- tral scafolds (M and 33) in CBS999.97(MAT1-2, M/33) porter genes, gene clusters for polyketide synthases underwent an unequal translocation to form two new (PKS), non-ribosomal peptide synthase (NRPS), and scafolds (F and X) in CBS999.97(MAT1-1, F/X) [8]. PKS–NRPS fusion proteins. Te QM6a-HiC annotation Like CBS999.97(MAT1-2, M/33), QM6a has a MAT1-2 (http://trichocode.com/index.php/t-reesei) became pub- mating-type locus and two ancestral scafolds M and 33. licly available in January, 2017 and the expectation values Due to chromosome heterozygosity and meiotic recom- (E) for the BLAST sequence alignments are not available, bination, sexual crossing of CBS999.97(MAT1-1, F/X) but claimed to be at least <e−75 [18]. with CBS999.97(MAT1-2, M/33) or QM6a often (>90%) Strictly speaking, the HiC draft genome is far from generates segmentally aneuploid (SAN) progeny [8]. Te equivalent to a complete genome sequence. We noticed CBS999.97 wild-isolate strain is also an excellent fungal that both the QM6a-v2.0 and QM6a-HiC drafts lack model for light responses since light variation greatly several evolutionarily conserved genes that are ubiqui- afects its sexual development and conidiation [7, 9, 10]. tously expressed in almost all studied eukaryotic organ- Constant light promotes conidiation and completely isms, including the recA family protein Rad51 and the inhibits stromata formation, whereas total darkness DNA repair protein Rad50. Terefore, we applied both causes a slowdown of the growth of stromata [10]. second- and third-generation sequencing (SGS and Te genomes of several Trichoderma species have TGS) platforms to resequence the QM6a genome. Te been sequenced and are publicly available from the single molecule real-time (SMRT) sequencing method Joint Genome Institute (JGI) of the US Department of developed by Pacifc BioSciences (PacBio) ofers much Energy. Te QM6a-v2.0 draft genome (33.4 Mb) contains longer reads of up to 60 kb [19]. After error correction 87 scafolds and 9129 predicted genes [11]. Te RUT- with short and high-quality Illumina MiSeq sequencing C30-v1.0 draft genome (32.7 Mb) has 182 scafolds and reads, the PacBio long reads were assembled into seven 9852 predicted genes [12]. Te genomes of Trichoderma telomere-to-telomere chromosomes. Our high-quality atroviride and Trichoderma virens were thought to be genome sequence provides a large quantity of new infor- larger than QM6a-v2.0, with sizes of 36.1 and 38.8 Mb, mation to facilitate functional and comparative studies of respectively, versus 34.1 Mb for QM6a, both encoding this industrially important workhorse fungus. more than 2000 additional predicted genes [4, 13]. Tese genomes have been used to identify key genes involved Results in some important biological processes [4, 13–15], e.g., Resequencing the QM6a genome the transcriptional factors and transporters that con- Te QM6a genome was resequenced using seven SMRT trol induction and expression of carbohydrate-active cells on the PacBio RSII platform. Following extrac- enzymes (CAZymes) and the plant cell wall degradation tion of reads with Trichoderma reesei-only sequences, enzymes. Using QM6a as a reference, it has also been we recovered the longest raw reads (≥16 kb) with up to reported that QM9414 and RUT-C30 might carry mul- 80× coverage, totaling 3,397,762,180 bp. Te hierarchi- tiple alternations, including rearrangements, point muta- cal genome assembly process program [20] was used to tions, insertions, and deletions [12, 16]. Recently, a group generate a preliminary PacBio draft with seven superscaf- of Trichoderma researchers have collectively annotated folds, a short unitig, and 1.8 kb of contaminating DNA and compared ~30% genes in the JGI genomes of T. ree- (Additional fle 1: Table A1). Tis short unitig was com- sei, T. atroviride, and T. virens [4]. pletely identical in nucleotide sequence to the QM9414 To reveal gene order and dynamic gene expression at mitochondrial genome (42,139 bp; NC_003388.1) [21], the chromosome level, a genome-wide chromosome indicating that the error rate for the preliminary PacBio conformation capture method (referred herein as “HiC”) draft was extremely low (<0.0024%). Li et al. Biotechnol Biofuels (2017) 10:170 Page 3 of 20 For error correction, the Illumina MiSeq 300 bp in QM6a-v2.0. Te most prominent assembly error paired-end reads (6.8 Gb) were collected and trimmed. was the frst and longest scafold, wherein the 5′ por- Reads (67.85%) with a quality score threshold (Q) greater tion (~2.46 Mb) was mapped to ChV and the 3′ portion than 30 were retained (Additional fle 1: Tables A2, A3). (~1.79 Mb) to ChIV (Additional fle 1: Table A7).